In an effort to further extend the number of targets for development of antiretroviral agents, we are investigating potential HIV integrase inhibitors using in vitro integrase assays. The active compounds are submitted for testing their antiviral activity in the NCI Anti-HIV Drug Screen. Conversely, we are testing active compounds from the screen for HIV- l integrase inhibition. The molecular interactions of drugs with the HIV- l integrase are investigated by comparing the drugs effects using several assays exploring different steps of the integration reaction: 1) DNA binding, 2) dinucleotide cleavage and differential use of attacking nucleophiles (water, 3'-DNA terminus, glycerol), 3) strand transfer (integration), 4) dis-integration (reverse of the integration reaction) by wild-type and truncated HIV- l integrases. We are also investigating the role of topoisomerases in integration and transcription. Our goals are to discover new antiviral agents, to determine whether drug screening of active agents will lead to the discovery of integrase inhibitors, to evaluate which step of the integration can be inhibited by drugs (enzyme oligomerization, DNA binding, 3'-processing, DNA strand transfer, disintegration), and to determine the drug binding site in the HIV- l integrase. In order to test the hypothesis that some of the inhibitors may interfere selectively with a specific functional domain of the enzyme (zinc finger, catalytic region, DNA binding domain), genetically altered HIV- l integrases are used. Discovery of new HIV integrase inhibitors may provide new strategies for antiretroviral therapy and basic knowledge for drug-enzyme interactions.